Pigmented Villonodular Synovitis (PVNS) of the Knee mimicking Septic Arthritis in a Paediatric Patient: A Case Report.

Pigmented villonodular synovitis (PVNS) is a benign but rare proliferative disorder of the synovium. It commonly occurs in the adult population and usually presents as a monoarticular disease. There are two types of PVNS, namely the localised and diffused type. The disease is often misdiagnosed due to its rarity especially in paediatric patients. Knee involvement in PVNS is the commonest form in children although other joints such as hip, foot, ankle, hip, sacroiliac joint and concurrent multiple joint involvements have also been reported. PVNS in paediatric patients is often misdiagnosed as septic arthritis, juvenile rheumatoid arthritis and bone sarcoma, and the diagnosis is usually often made late due to its vague presentation. The majority of PVNS cases are managed by surgery either via open or arthroscopic synovectomy except in a few paediatric patients as described in the literature. This case report of PVNS is of a knee in 11-year-old boy who was initially treated as septic arthritis. The synovium appearance mimicked the features of PVNS during a knee arthrotomy washout, and histopathological examination confirmed the diagnosis. The knee symptoms had significantly improved without additional surgery, and good functional knee motion was achieved, with no sign of recurrence, after two years of follow-up.

Pigmented Villonodular Synovitis (PVNS) of the Knee mimicking Septic Arthritis in a Paediatric Patient: A Case Report

@#Pigmented villonodular synovitis (PVNS) is a benign but rare proliferative disorder of the synovium. It commonly occurs in the adult population and usually presents as a monoarticular disease. There are two types of PVNS, namely the localised and diffused type. The disease is often misdiagnosed due to its rarity especially in paediatric patients. Knee involvement in PVNS is the commonest form in children although other joints such as hip, foot, ankle, hip, sacroiliac joint and concurrent multiple joint involvements have also been reported. PVNS in paediatric patients is often misdiagnosed as septic arthritis, juvenile rheumatoid arthritis and bone sarcoma, and the diagnosis is usually often made late due to its vague presentation. The majority of PVNS cases are managed by surgery either via open or arthroscopic synovectomy except in a few paediatric patients as described in the literature. This case report of PVNS is of a knee in 11- year-old boy who was initially treated as septic arthritis. The synovium appearance mimicked the features of PVNS during a knee arthrotomy washout, and histopathological examination confirmed the diagnosis. The knee symptoms had significantly improved without additional surgery, and good functional knee motion was achieved, with no sign of recurrence, after two years of follow-up.

A Descriptive Mixed-Methods Analysis of Sexual Behavior and Knowledge in Very Young Children Assessed for Sexual Abuse: The ASAC Study.

Child sexual abuse (CSA) is a worldwide problem with serious consequences. We hypothesized that worrisome sexual behavior and knowledge would frequently be reported in children assessed after CSA. We therefore investigated (A) what types of sexual behaviors and knowledge were reported by parents of young children assessed for CSA; (B) in what cases such behaviors and knowledge were worrisome; and (C) how such children responded verbally and non-verbally during child interviews. We conducted a mixed-methods study, including qualitative inductive content analysis and quantitative analysis. It included 125 children (76 boys, 60.8%; median age 3.3 years, age range 0-11), all involved in the Amsterdam sexual abuse case (ASAC) and examined for highly suspected (n = 71) or confirmed CSA (n = 54). We identified themes from (1) the parent reports: sexual behavior (e.g., self-stimulation, touching others, imitation of sexual acts), fears and anxiety with regard to sexuality, and sexual utterances (sexual slang, references to sexual acts); and (2) the child interviews: behavioral reactions (avoidance, distractive behaviors), emotional reactions (anger, aggression), and verbal reactions (conspicuous utterances, refusal to talk about specific subjects). In 37% of the children the sexual behavior was deemed worrisome or very worrisome. Clinicians who assess children for CSA are advised to focus in particular on sexual behavior problems and inappropriate sexual knowledge.

Psychosocial symptoms in very young children assessed for sexual abuse: A qualitative analysis from the ASAC study.

Child sexual abuse (CSA) is a worldwide problem with serious consequences. No recognizable symptom pattern for suspected CSA has yet been identified in very young children. We aim to investigate psychosocial symptoms in a sample of children with confirmed or strongly suspected CSA and the interpretations given to such symptoms by independent clinical experts. Secondly we examined whether experts were able to identify confirmed victims of severe CSA. A qualitative study including inductive content analysis of medical files and focus group discussions with independent experts on the interpretation of psychosocial symptoms was conducted. We included 125 children (76 boys, 60.8%, and 49 girls, 39.2%; median age 3.3 years; age range 0-11) who were involved in the Amsterdam sexual abuse case (ASAC) and had been examined for strongly suspected CSA. We identified four themes among the psychosocial symptoms: problems concerning emotions, behavior, toilet training, and development, whether or not associated with the daycare center or the perpetrator. Clinical experts identified signs of posttraumatic stress disorder (PTSD), regression in continence skills (not otherwise explained), and problems triggered by exposure to the perpetrator or the abuse location as concerning symptoms for CSA. Less concerning symptoms were designated as worrisome if they were numerous and there was no clear explanation for these symptoms. A clear symptom pattern was lacking and about half of the confirmed severe victims of CSA did not display any psychosocial problems. Therefore, it is difficult for experts to identify confirmed CSA victims. Thus, the assessment of suspected CSA should be over time and multidisciplinary.

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding.

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.

A paramagnetic species with unique EPR characteristics in the active site of heterodisulfide reductase from methanogenic archaea.

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron-sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and coenzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-electron step with a midpoint-potential of -185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe-enriched enzyme indicates that it is at least partially iron based. The g values (gxyz = 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe-2S]+, [3Fe-4S]+, [4Fe-4S]+ or [4Fe-4S]3+ cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H-S-CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe-4S]3+ cluster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.

Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II.

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.

Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H2 value.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium pasteurianum ferredoxin.

The [2Fe-2S] cluster of the ferredoxin from Clostridium pasteurianum is coordinated by cysteines 11, 56, and 60 and by a fourth cysteine, residue 24 in the wild-type protein, located on a flexible and deletable loop around residues 14-30. New mutated forms of this ferredoxin show that the fourth cysteine ligand can be located in any one of positions 14, 16, 21, 24, or 26. Another set of molecular variants has unveiled a new case of ligand swapping on the cysteine 60 ligand site. Replacement of cysteine 60 by alanine and introduction of a cysteine in position 21 yielded a ferredoxin that assembles a [2Fe-2S] cluster of which the ligands are cysteines 11, 21, 24, and 56. This cysteine ligand pattern is similar to that occurring in plant-type or mammalian-type ferredoxins, although the overall sequence similarities are below detection. Moreover, the vibrational and electronic properties of the resulting [2Fe-2S]2+/+ center, as revealed by resonance Raman and EPR studies, are strikingly similar to those of mammalian-type ferredoxins. The extensive set of mutated forms of the C. pasteurianum ferredoxin now available indicates that cysteine ligand exchange may occur on residues 24 and 60, but not on residues 11 and 56. It is thus suggested that cysteines 24 and 60 are part of a solvent accessible aspect of the Fe-S cluster, whereas cysteines 11 and 56 are buried and form the more rigid part of the polypeptide ligand framework. In view of the unprecedented versatility of this [2Fe-2S] cluster and of its polypeptidic environment, the introduction of ligands other than cysteine in various positions has been attempted. These experiments have remained unsuccessful, and even including previous studies, noncysteinyl ligation has been obtained with this protein in only very few cases. The data provide an extensive confirmation that Fe-S clusters have a strong preference for thiolate ligation and rationalize the relatively rare occurrence of noncysteinyl ligation in native Fe-S proteins.

Interactions between P-glycoprotein substrates and other cationic drugs at the hepatic excretory level.

1. In the present study it was tested whether known P-glycoprotein (P-gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tri-n-butylmethylammonium (TBuMA) (a relatively small type 1 organic cation), rocuronium (Roc) (a bulky type 2 organic cation) and the classical P-gp substrate doxorubicin (Dox). 2. Inhibitors were given in a 4 fold molar excess to the substrate studied. To minimize an interaction of the substrates at the hepatic uptake level, the competing compounds were added when over 55% to 85% of the administered dose of the model compounds had been removed from the perfusate and taken up by the liver. 3. We found a mutual interaction between TBuMA and procainamidethobromide (PAEB), both type 1 cationic compounds during biliary excretion. Interestingly, type 2 compounds, such as rocuronium, clearly inhibited type 1 cationic drugs as well as Dox secretion into bile, whereas type 1 compounds did not significantly inhibit type 2 drug excretion into bile. The type 1 cations PAEB and TBuMA only moderately inhibited Dox biliary excretion. Dox did not inhibit the biliary excretion of the type 2 agent rocuronium whereas rocuronium reduced Dox biliary excretion by 50% compared to controls. 4. MDR substrates/reversal agents like verapamil, quinine, quinidine and vinblastine strongly reduced both type 1 and type 2 organic cation excretion into bile. Dox secretion into bile was also profoundly reduced by these drugs, vinblastine being the most potent inhibitor in general. 5. The lack of mutual inhibition observed in some combinations of substrates may indicate that major differences in affinity of the substrates for a single excretory system exist. Alternatively, multiple organic cation transport systems with separate substrate specificities may be involved in the biliary excretion of amphiphilic drugs. Furthermore, the present study revealed a clear positive correlation between the lipophilicity of the potential inhibitors studied and their respective inhibitory activity on the biliary excretion of the model drugs investigated. 6. Our data are compatible with a potential involvement of P-glycoprotein in the hepatobiliary excretion of doxorubicin as well as of some type 1 and type 2 organic cations. Furthermore we postulate that the hydrophobic properties of the amphiphilic cationic drugs studied play a crucial role in the accommodation of these agents by P-glycoprotein and/or other potential cationic drug carrier proteins in the canalicular membrane.

[2Fe-2S] to [4Fe-4S] cluster conversion in Escherichia coli biotin synthase.

The type and properties of the Fe-S cluster in recombinant Escherichia coli biotin synthase have been investigated in as-prepared and dithionite-reduced samples using the combination of UV-visible absorption and variable-temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman spectroscopies. The results confirm the presence of one S = 0 [2Fe-2S]2+ cluster in each subunit of the homodimer in aerobically purified samples, and the Fe-S stretching frequencies suggest incomplete cysteinyl-S coordination. However, absorption and resonance Raman studies show that anaerobic reduction with dithionite in the presence of 60% (v/v) ethylene glycol or glycerol results in near-stoichiometric conversion of two [2Fe-2S]2+ clusters to form one S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination. The stoichiometry and ability to effect reductive cluster conversion without the addition of iron or sulfide suggest that the [4Fe-4S]2+ cluster is formed at the subunit interface via reductive dimerization of [2Fe-2S]2+ clusters. EPR and VTMCD studies indicate that more than 50% of the Fe is present as [4Fe-4S]+ clusters in samples treated with 60% (v/v) glycerol after prolonged dithionite reduction. The [4Fe-4S]+ cluster exists as a mixed spin system with S = 1/2 (g = 2. 044, 1.944, 1.914) and S = 3/2 (g = 5.6 resonance) ground states. Subunit-bridging [4Fe-4S]2+,+ clusters, that can undergo oxidative degradation to [2Fe-2S]2+ clusters during purification, are proposed to be a common feature of Fe-S enzymes that require S-adenosylmethionine and function by radical mechanisms involving the homolytic cleavage of C-H or C-C bonds, i.e., biotin synthase, anaerobic ribonucleotide reductase, pyruvate formate lyase, lysine 2, 3-aminomutase, and lipoic acid synthase. The most likely role for the [4Fe-4S]2+,+ cluster lies in initiating the radical mechanism by directly or indirectly facilitating reductive one-electron cleavage of S-adenosylmethionine to form methionine and the 5'-deoxyadenosyl radical. It is further suggested that oxidative cluster conversion to [2Fe-2S]2+ clusters may play a physiological role in these radical enzymes, by providing a method of regulating enzyme activity in response to oxidative stress, without irreversible cluster degradation.

Changes in the electronic structure around Ni in oxidized and reduced selenium-containing hydrogenases from Methanococcus voltae.

The selenium-containing F420-reducing hydrogenase from Methanococcus voltae was anaerobically purified to a specific hydrogen-uptake activity of 350 U/mg protein as determined with the natural electron acceptor. The concentrated enzyme was used for EPR-spectroscopic investigations. As isolated, the enzyme showed an EPR spectrum with g(xyz) values of 2.21, 2.15 and 2.01. Illumination of such samples at low temperatures led to an EPR spectrum with g(xyz) values of 2.05, 2.11 and 2.29. These spectra are typical for [NiFe]hydrogenases in the active state. Spectra of samples enriched in 77Se showed a hyperfine interaction between the unpaired spin of the nickel ion and the nuclear spin of one 77Se atom before and after illumination. A 90 degree flip of the electronic z-axis is proposed to explain the hyperfine interaction in both states. This has been demonstrated previously only for the F420-non-reducing hydrogenase from M. voltae, where the selenium atom is present as a selenocysteine residue on an unusually small separate subunit [Sorgenfrei, O., Klein, A. & Albracht, S. P. J. (1993) FEBS Lett. 332, 291-297]. The results demonstrate that the three-dimensional structures of the active sites in the selenium-containing F420-reducing and F420-non-reducing hydrogenases from M. voltae are highly similar and hence are not influenced by the unusual subunit structure of the latter enzyme. Oxidized samples containing either natural selenium or 77Se were prepared from the F420-reducing and the selenium-containing F420-non-reducing hydrogenase. Both enzymes exhibited EPR spectra typical for [NiFe]hydrogenases in the inactive 'ready' state. In contrast to the reduced form, no splitting of the nickel-derived signal due to the nuclear spin of 77Se was observed in the oxidized state, indicating that the electronic z-axis is perpendicular to the Ni-Se direction.

Interactions of 77Se and 13CO with nickel in the active site of active F420-nonreducing hydrogenase from Methanococcus voltae.

The selenium-containing F420-nonreducing hydrogenase from Methanococcus voltae was prepared in the Nia(I) middle dotCO state. The effect of illumination on this light-sensitive species was studied. EPR studies were carried out with enzyme containing natural selenium or with enzyme enriched in 77Se. Samples were prepared with either CO or 13CO. In the Nia(I) middle dotCO state, the nuclear spins of both 77Se (I = 1/2) and 13C (I = 1/2) interacted with the nickel-based unpaired electron, suggesting that they are positioned on opposite sites of the nickel ion. In the light-induced signal, the interaction with 13CO was lost. The 77Se nuclear spin introduced an anisotropic hyperfine splitting in both the dark and light-induced EPR signals. The data on the active enzyme of M. voltae are difficult to reconcile with the crystal structure of the inactive hydrogenase of Desulfovibrio gigas (Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587) and suggest a structural change in the active site upon activation of the enzyme.

Infrared-detectable groups sense changes in charge density on the nickel center in hydrogenase from Chromatium vinosum.

Fourier transform infrared studies of nickel hydrogenase from Chromatium vinosum reveal the presence of a set of three absorption bands in the 2100-1900 cm-1 spectral region. These bands, which do not arise from carbon monoxide, have line widths and intensities rivaling those of a band arising from the carbon monoxide stretching frequency (v(CO)) in the Ni(II).CO species of this enzyme [Bagley, K. A., Van Garderen, C. J., Chen, M., Duin, E. C., Albracht, S. P. J., & Woodruff, W. H. (1994) Biochemistry 33, 9229-9236]. The positions of each of these three infrared absorption bands respond in a consistent way to changes in the formal redox state of the nickel center and to the photodissociation of hydrogen bound to the nickel. Up to eight different states of the nickel center have been produced, depending on the redox state and/or the activity state of the enzyme and the presence of carbon monoxide. In seven of these states, the three IR absorption bands in the set have unique frequency positions. It is concluded that the set is due to intrinsic, non-protein groups in the enzyme, whose identities are presently unknown, and that these groups are situated very close to the nickel center and sense the charge density at the Ni site.

Infrared studies on the interaction of carbon monoxide with divalent nickel in hydrogenase from Chromatium vinosum.

Infrared spectra of a carbon monoxy-bound form of the EPR silent Ni(II) species of hydrogenase isolated from Chromatium vinosum are presented. These spectra show a band at 2060 cm-1 due to v(CO) for a metal-CO complex. This absorbance shifts to 2017 cm-1 upon exposure of the enzyme to 13CO. This band is attributed to v(CO) from a Ni(II)-CO species. It is shown that the CO on this species is photolabile at cryogenic temperatures but rebinds to form the original carbon monoxy species at temperatures above 200 K. In addition to the v(CO) band, infrared lines are detected at 2082, 2069, and 1929 cm-1, which shift slightly higher in frequency upon photolysis of the CO from the Ni. These infrared bands do not arise from CO itself on the basis of the fact that the frequency of these bands is unaffected by exposure of the enzyme to 13CO. Experiments in D2O show that these bands do not arise from an exchangeable hydrogen species. It is concluded that these non-CO bands arise from species near or coordinated to the Ni active site. The possible nature of these bands is discussed.

Further characterization of the spin coupling observed in oxidized hydrogenase from Chromatium vinosum. A Mössbauer and multifrequency EPR study.

Hydrogenase from Chromatium vinosum contains 1 Ni, 11-12 Fe, and ca. 9 sulfides. EPR and Mössbauer studies of the enzyme prepared in four different oxidation states show that the enzyme contains two Fe4S4 and one Fe3S4 cluster. In the oxidized (2+) state, the Mössbauer parameters of the two Fe4S4 clusters are typical for this cluster type. Upon reduction, however, these clusters do not exhibit the familiar g = 1.94 signal. The unusual nature of the reduced clusters is also borne out by the Mössbauer spectra which exhibit fairly small magnetic hyperfine interactions similar to those of centers I and II of the Desulfovibrio gigas enzyme. The Mössbauer spectra of the Fe3S4 cluster in the oxidized (1+) and reduced states are typical for this cluster type. The C. vinosum hydrogenase undergoes a reversible redox reaction at Em = +150 mV (vs NHE). Above +150 mV the EPR spectra exhibit signals (previously called signals 2 and 4) that reflect a weak interaction between Ni(III) and an Fe-containing moiety. By clamping the Ni in the diamagnetic Ni(II).CO form, we have discovered that signal 2 (X-band resonances at g = 2.01, 1.974, and 1.963) involves the Fe3S4 cluster and an as yet unidentified paramagnetic moiety. The "coupled" system exhibits magnetic hyperfine interactions quite different from those of the uncoupled [Fe3S4]1+ cluster. We have not yet been able to assign a spin to the coupled state but some of the features of the state are reminiscent of an S = 1 system. The Mössbauer data suggest, but do not prove, that an extra Fe site may be present that shuttles between low-spin Fe(III) and low-spin Fe(II) with Em = +150 mV. The Fe(III) may be located between the Ni(III) and the Fe3S4 cluster enabling it to mediate the interaction between the cluster and the Ni site. In this picture, the Fe(III) site is part of the coupled state that gives rise to signal 2. Other possibilities for signal 2 involve a ligand-based oxidation of the [Fe3S4]1+ cluster or generation of a nearby radical.